The intent of this project is to develop new and to improve existing biophysical methodology for the characterization of biological macromolecules in solution, and to apply these methods collaboratively to the study of proteins and their interactions. Experimental techniques employed are analytical ultracentrifugation, static and dynamic light scattering, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance biosensing. For the study of reversible formation of multi-protein complexes in solution, we have developed a novel multi-signal analysis for sedimentation velocity analytical ultracentrifugation. It permits the label-free detection of sedimentation coefficient distributions of currently up to three protein components, and thus measure the stoichiometry of multiple hydrodynamically separated protein complexes. We have also analyzed the influence of binding kinetics on the time-scale of sedimentation. Studies were conducted for the proof of principle, followed by first model applications to different biological systems of interacting proteins. These include the collaborative study of the extended hetero-association of plasminogen activator inhibitor (PAI-1) complexes with vitronectin, which is important in redirecting the localization of vitronectin from the circulation to the extracellular matrix where an adhesive function is manifested. The multi-signal sedimentation velocity method has been used to document the occupation of two PAI-1 distinct binding sites on vitronectin as a requisite for the assembly of higher-order complexes. In the collaborative application to the study of a signal transduction complex after T-cell activation, formation of extended triple protein complexes of adaptor proteins was detected. We have collaboratively applied the previously developed techniques hydrodynamic and thermodynamic sedimentation techniques to the study of PH domain of ArfGAPs, the HIV envelope proteins and their interactions, interactions of NK (natural killer) cell surface receptors, malarial surface proteins, clathrin basket assembly, cryptophycin induced tubulin structures, iron regulatory protein 1 and 2 interactions with IREs, and the glycolytic enzyme gucocerebrosidase and its interactions with alpha synuclein (the causative agent in Parkinson?s disease). We have continued our efforts to collaboratively apply our biophysical methodology to the characterization of the filamentation process of alpha synuclein, which is associated with Parkinsons disease. In the area of optical biosensing, progress was made in the computational approach to determine the functional heterogeneity of surface binding sites. The previously introduced model was extended to account for mass transport limited surface binding. This was applied to antibody-antigen model systems, followed by the analysis of integrin receptors to a variety of ligands relevant to leukocyte interaction with tissue proteins, the study of antibodies to anthrax protective antigen, and the study of MAPK-kinases with anthrax LF protein. We have also developed techniques for studying small molecule binding with surface-immobilized nucleic acid and proteins. A first application was the study of inhibitors of HIV reverse transcriptase and human RNAse H to aid in the screening of compound libraries of potential drug candidate molecules, and to analyze the mechanism of the inhibition. A further methodological development is aimed at increasing the recovery of surface captured molecules for interfacing biosensors with mass spectroscopy.